Sexual hormones and method of preparing them



Patented A r. 4, 1939 i 02,152,626

UNITED STATES PATENT" oFrIc-s SEXUAL HORMONES AND METHOD or ran mama THEM Wilhelm Dirscherl, Frankfort-on-the-Main, and Fritz. Hanusch, Heidelberg, Germany, assignors to Bare Chemicals Inc., Nepera Park, N. Y., a corporation of New York No Drawing. Original application November 23,

1936, Serial No. 112,350. Divided and. this application August 19, 1937, Serial No. 159.930. In Germany November 25, 1935 17 Claims. (01. 260-397) This application is a division of our copending methanol, formic aldehyde, sulfurou's acid and ,patent application Ser. No. 112,350, filed Nov. 23, so on, V r r 1936. The solvent is now removed bvdistilling, f r

Our invention relates to substances acting as instance, after neutralizing the mixture, prefersexual hormones and especially. to substances ably in vacuo. The remaining mixture of sub- 5 having the activity of a male sexual hormone as stances is separated from inorganic components well as of a corpus luteum hormone. It deals by means of water, whilst the organic compoespecially with the manner of preparing such nents containing the active substances are taken substances in anew and more emcient way than 'up by a suitable organic solvent e. g. ether.

- it was possible up till now. Other objects will For removing inactive organic substances of 10 hereinafter appear. acid reaction, the solution is'now treated, suit- We have found that hormone likeacting subably in the presence of water, by neutralising stances can be obtained by treating allocholesagents as for instance caustic alkali, alkaline terol, or raw materials containing this substance earth, ferric hydrate and so o'n'and by eliminatin, the acid or neutral medium by oxidizing ing the salts thus obtained. Afterwards the in- 15 agents. The substances are prepared by sepaactive volatile components are distilled in vacuo rating after the oxidation the reacting mixture or with a suitable vapor as a carrier as for infrom inefficient, acid and volatile components stance steam. In somecases the sequence of reand from unchanged initial material and by conmoving the acid and volatile reaction compocentrating, purifying and isolating in the mannents may also be reversed. The product thus 20 ner usual in preparing sexual hormones. obtained, a brownish oil, shows a strong male Oxidation may be carried out by oxidizing I hormone lik'eactivity and at the same time the agents especially vigorously acting oxidizing activity of a corpus luteum hormone. agents, particularly by metal compounds rich in For. further purificatiominactive substances,

oxygen as e. g. chromic acid also called chromic if any, especially unattacked initial material, are 5 anhydride, permanganic acid, acetate of tetraseparated from the active substances... This may valent lead (Pb(OCOCH3)4), further by acids be carriedout preferably bythe following ways: which are derived from hydrogen peroxide as 1. By separation by means of fractional cryse. g. peracetic acid. Concentrated nitric acid is tallisation from suitable solvents preferably at not suitable as an oxidizing agent. The reaction low, temperatures; I 4 v is carried out preferably in the presence of dilu- 2. By treating with adsorption agents as e. g. ents especially of organic character, as e. g. glaalumina or bleaching clay; by acting in this mancial acetic acid or strong acetic acid. Oxidation ner the hormone acting substance remains as a by electrolysis may be taken into account, the rule fixed on the adsorption agent whilst the inimetal compounds mentioned above acting as tial material is substantially removed without 35 oxygen transporters. any difilculty by a thorough washing;

Oxidation may be executed at room tempera- 3. By distributing .amongst two solvents as ture as well as at higher temperatures, prefere. g. ligroin and an aqueous alcohol; ably at temperatures between 50-'70 C.,tempera- 4. By treating with ketone reagents as e. g. tures between 70-18%? C., and by special condihydroxylamine, hydrazines, especially simple or 40 tions e. g. in case of a suitable dilution or a'very substituted phenyl hydrazines, semicarbazide, short reaction time, temperatures above 100 C. derivatives of aminoacet-hydrazide and so on, the may also be used. Particularly the oxidation by addition or condensation compounds thus formed chromic acid requires higher temperatures for being separated by one of the methods 1-3. example of about C. and even higher; but Especially commendable are combinations of 45 this feature does not exclude'a beginning at lower the said methods particularly the combination of temperatures e. g. at room temperature. It is fractional crystallisation together with following recommendable to take care of a good thorough adsorption. mixing of the oxidizing agent with the reaction After removal of the initial material particumixture containing the substance to be oxidized larly on working by adsorption methods, the 9.0- 50 which can take place by stirring or eventually by tive substances showing a ketone character may adding emulsifying substances. be obtained by distillation or sublimation in high After having finished the oxidation, an excess vacuo and in some cases by subsequent recrysof the oxidant, if any, is destroyed by means of tallisation or by combination of the three methsubstances easily oxidisable, as for example ods. A product is thus obtained which shows at 55 2 the same time .the activity of a male sexual hormone and of a corpus luteum hormone. By repeated application of the mentioned purification methods, as, for instance, sublimation in prepare first the compounds with ketone re-- agents. Substances giving colored compounds as e. g. nitro-phenylhydrazines are particularly suitable reagents because the reaction mixture thus obtained-can .be .divided by means of the chro- -matographical adsorption method by Tswett (see:

Berichte d. deutschen botan. Gesellschaft", vol.

24 (1906) page 234, further, the monograph of 'Palmer: Carotinoids and Related Pigments, New

. York; The Chemical Catalog Go. (1922)) in frac-.

' ponent.

tions containing a male hormone active component and a corpus luteum hormone active com- Affer liberating the adsorbed compounds by eluting liquids as e. g. alcohols, ether,

chloroform, acetone, dioxane, and splitting oil the ketone reagents, the pure substances may be obtained by the above mentioned methods of distillation and sublimation in high vacuo and in some cases by recrystallisation. The working up by meansof reagents giving colored compounds in connection with the chromatographical adsorption method following afterwards may be applied also when the initial material is not yet or only partly removed. In these cases several colored adsorptionzones may be stated according to which unchanged initial material and other inefl'icient reaction components may be separated from the active substances and in some cases also the male. acting from the corpus luteum hormone acting substance.

The decomposition of the compounds formed of the active substances and .the ketone reagents. is carried out in .the usual manner by dilute acids, particularly by dilute oxalic acid, whereas the subsequent purification of the active product thus obtained is efiected for instance by sublimation in high vacuo as above described.

E:tampZe.20 'g. allocholesterol are dissolved in 1000 cc. glacial acetic acid. Into this solution are dropped at room temperature with stirring a solution of 37 g. chromic acid in 180v cc. acetic acid (about 90%). Alter having been added about 4 g. chromic acid, the temperature of the reaction mixture is increased to about 50 0. this temperature being. maintained till the rest of the chromic acid has reacted. After the oxidation an excess of chromic acid, it any, is destroyed by addition of methanol and the glacial acetic acid is distilled in vacuo; the remaining reaction mixture is taken up with water and shaken several times with ether. The ethereal extract is washed with water, with a dilute solution of caustic soda and once more with water; the etheris then removed by distillation. The resl-' due is now freed from volatile substances by steam-distillation and the remainder shaken out with. ether. The ethereal solution is dried with sodium sulfate and freed from ether by distilling. The remaining product is a brownish oil, soluble in many organic solvents e. g. alcohols. chloroform, ether, benzene, ligroin, sesame oil, glacial luteum hormone. The amountof the hormonal activity depends on the quantity of inactive byproducts particularly of unconverted initial materiaL- Asa rule the capons comb unit according to Fussganger (see: Med. und chem. Abhandlungen der med. chem. Forschungsstatten der I. G.

about 150-300 mg. Products of less and some-- times of more activity may also be obtained.

By removing the inactive byproducts especially the allocholesterol still present the activities can be attained a five fold, a ten fold and even a higher value and they can be-approximated to the acetic acid, slightly soluble in water, having the activity ofa male sexual hormone and of a corpus values of pure'male sexual hormone substances and pure corpus luteum hormone substances.

For attaining this further purification, several methods may be applied. For instance the prodnot is allowed to cool in a refrigerator, whereby the main part of the initial material forms crystals which may be separated by sucking them off. The filtrate is diluted with alcohol; an alcoholic male sexual hormone andafraction of a corpus luteum hormone.

Instead of purifying by means of ketone re-" agents, a further treatment may also be carried outby adsorption methods while the mainpart of unchanged allocholesterol has been removed by refrigerating. On applying alumina or bleaching clay.- inactive substances particularly the. initial material are not adsorbed or only weakly ad-. sorbed so that they can be removed by washing out with the same solvent in which the product is dissolved, whilst the active substances remain adsorbed and can be removed by special wash-.

ing liquids. The mixture of substances dissolved in benzene, toluene, xylene or a similar solvent is for instance adsorbed on a column ofalumina of loose texture filled e. g. in a wide glass tube. Benzene or one of the other mentioned solvents is sucked through the adsorption mixture until the filtrate substantially leaves on evaporating no residue of inactive substances. The active substances are now liberated from the adsorbent by suitable eluting liquids as for instance alcohols,

particularly methanol, ether, chloroform, acetone. The product remaining after evaporating of the eluting liquid may be decomposed by means of a dilute acid and the "active substance further treated as above described.

When using as ketone reagents substances yielding colored compounds, as e. g. 2.4-dinitrophenylhydrazine, the iormed dinitrophenylhydrazones may be dissolved in a suitable solvent and divided infractions by the chromatographical adsorption method. To this end the solution rated by developing into different colored zones.

' liberated decomposed by a dilute acid and further By sectioning the column according to the colored zones the inactive substances can be separated from the active substances and in some cases also the substance acting as a male sexual hormone from the substance acting as a corpus luteum hormone.

The several parts of the column are then treated with suitable eluents, the substances thus treated for instance by sublimation in high vacuo and fractional recrystallisation as above mentioned.

Various changes may be made in the details disclosed in the foregoing specification without departing from the invention or sacrificing the advantages thereof.

' which comprises treating a mixture of substances containing allocholesterol by vigorously acting oxidizing agents consisting of the'group of oxygen compounds of metals containing large amounts of available oxygen, of tetravalent-lead and of acids .derived from hydrogen peroxide and separating the produced active substance from the reaction mixture.

3. The process of producing hormone like substances having the activity oi'. a male sexual hormone as well as of a corpus luteum hormone which comprises treating allocholesterol by vigorously acting oxidizing agents consisting of the group of oxygen compounds of metals containing large amounts of available oxygen, of tetravalent lead and of acids derived from hydrogen peroxide and separating the produced active substance from the reaction mixture.

4. In the process of claim 1 the oxidation carried out between room temperature and C.

5. The process of producing hormone like substances having the activity of a male sexual hormone as well as of a corpus luteum hormone which comprises treating allocholesterol byv chromic acid as an'oxidizing agent at a tempera ture of about 50 C.

6. The process of producing hormone like substances having the activity of a male sexual hormone as well as of a corpus luteum hormone which comprises treating a mixture of substances containing allocholesterol by vigorously acting oxidizing agents consisting of the group of oxygen compounds of metals containing large amounts of available oxygen, of tetravalent lead and of acids derived from hydrogen peroxide, removing from the reaction mixture the acid components. by neutralising agents, the volatile components by distillation and separating the produced active substances from the reaction mixture.

.7. The process of producing hormone like substances having the activity of a male sexual hormone as well as of a corpus luteum hormone which comprises treating a mixture-containing duced active substances from the reaction mixture.

8. The process of producing hormone likev substances having the activity of a male sexual hormone as well as of a corpus luteum hormone which comprises treating a mixture containing allocholesterol by vigorously acting, oxidizing agents consisting of the group oi? oxygen compounds of metals containing large amounts of available oxygen, of tetravalent lead and of acids derived from hydrogen peroxide, removing from the reaction mixture the acid components by neutralising agents, the volatile components by distillation and separating the produced active substances from the reaction mixture by fractional. crystallisation at low temperatures.

9. The process of producing hormone like substances having the activity of a male sexual hormone as well as of a corpus luteum hormone which comprises treating a mixture containing allocholesterol by-vigorously acting, oxidizing agents consisting of the group of oxygen compounds of metals containing large amounts of available oxygen, of tetravalent lead and of acids derived from hydrogen peroxide; removing.

from the reaction mixture the acid components by neutralising agents, the volatile components by distillation'and separating the produced active substances from the reaction mixture by adsorption agents.

10-. The process of producing hormone like substances having the activity of a male sexualhormone as well as of a corpus luteum hormone which comprises treating a mixture containing allocholesterol by vigorously acting, oxidizing 'by distillation, treating the substance thus obtained with an adsorption agent and separating the active substances fromthe reaction mixture remaining adsorbed, by eluting liquids.

11. The process of producing hormone like substances having the activity of a male sexual hormone as well as of a corpus luteum hormone which comprises treating a mixture containing allocholesterol. by vigorously acting, oxidizing agents consisting of the group of oxygen compounds. of metals containing large amounts 01' available oxygen, of tetravalent lead and of acids derived from hydrogen peroxide, removing from the reaction mixture the acid components by neutralising agents, the volatile components by distillation and treating the product thus obtained with ketone reagents.

12. The process of producing hormone like substances having the activity of a male sexual hormone as well as of a corpus luteum hormone which comprises treating a mixture containing allocholesterol by vigorously acting, oxidizing removing agents consisting of the group of oxygen compounds of metals containing large amounts of available oxygen, .of tetravalent lead and of acids derived from hydrogen peroxide, removing the acid components by neutralising agents, the volatile componentsby distillation, treating the product thus obtained with ketone reagents, isolating the formed addition product, and obtaining the active substance by decomposing the adthe produced active substance, from the reaction mixture by fractional crystallisation at low temperatures, treating the product thus obtained with ketone reagents, isolating the resulting compound, decomposing it by a dilute acid and further purifying the active substances by sublimation in high vacuo and subsequent recrystallisation.

14. The process of producing hormone likesubstances which comprises treating a mixture containing allocholesterol by vigorously acting oxidizing agents consisting of the group of oxygen,

compounds of metals containing large amounts of available oxygen, of tetravalent lead and of acids derived from hydrogen peroxide, removing the acid components by neutralising agents, the volatile components by distillation, separating the produced active substance from the reaction mixture by fractional crystallisation at low temperatures, treating the product thus obtained with ketone reagents, isolating the resulting compound, decomposing it by a dilute acid and dividing the active substance into two fractions one having the activity of a male sexual hormone and the other the activity of a corpus luteum hormone. p

15. The process of producing hormone like substances which comprises treating a mixture containing allocholesterol by vigorously' acting oxidizing'agents consisting of the group of oxygen compounds of metals containing large amounts of available oxygen, of tetravalent lead and of acids derived from hydrogen peroxide, removing the acid components by neutralising agents, the volatile components by distillation, separating the produced active substance from the reaction mixture by fractionalcrystallisation at low temperatures, treating the product thus obtained with ketone reagents, isolating the resulting compound, decomposing it by a dilute acid and dividing by sublimation in a high vacuo' and subsequent recrystallisation the active substance .into two fractions one having the activity of a male sexual hormone and the other the activity of a corpus luteum hormone. h

16. The process of producing hormone like substances having the activity of a male sexual" hormone as well as of a corpusluteum hormone which comprises treating a mixture containing allocholesterol by vigorously acting oxidizing agents consisting of the group of oxygen compounds of metals containing large amounts of acid components by neutralising agents, the volatile components by distillation, separating the produced active substance from the main part of the mixture by fractional crystallisation at low temperatures, treating the product thus obtained with ketone reagents yielding colored compounds, adsorbing the formed compound on a suitable adsorbant and separating by developing, according 'to the chromatographical adsorptiv'e method, the

active substance from the reaction mixture.

17. A new hormone product, an oxidation prodnot of allocholesterol, a brownish oil of ketone character, soluble in alcohols, chloroform, ether, glacial acetic acid, benzene, ligroin, sesame oil,

slightly soluble in water, having the activity of a male sexual hormone on the capons comb and on the seminal vesicle of the mouse as well as of a corpus luteum hormone.

WILHELM DIRSCHERL. FRITZ HANUSCH.

-available oxygen, of tetravalent leadand of acids derived from hydrogen peroxide, removing the 

